Human carboxylesterase 1 (hCES1) is the most abundant hepatic hydrolase and is encoded by two CES1 genes, i.e. CES1A1 and CES1A2. hCES1 plays a prominent role in the hydrolysis of numerous structurally diverse carboxylic acid esters, carbamates, thioesters, and amide compounds. hCES1-mediated hydrolysis typically results in drug deactivation yet alternatively, it serves to activate many ester-based prodrugs, which include but not limited to oseltamivir (Tamiflu(R)), trandolapril, imidapril, cilazapril, enalapril, quinapril, delapril, temocapril, irinotecan, and lovastatin. These ester-based prodrugs require activation through hCES1 catalyzed hydrolysis to release their respective active metabolites to produce the intended pharmacological effects. Recently, several CES1 genetic variants have been discovered in our laboratory and others. Markedly altered pharmacokinetic and pharmacodynamic profiles of the hCES1 selective substrate methylphenidate have been observed in a human subject carrying the hCES1 variants, Gly143Glu and Asp260fs, during our preliminary studies. Other reports of deficient hCES1 activity by other research groups in research subjects have followed. Additionally, our preliminary studies of these two variants have found them to exhibit markedly decreased enzymatic activity towards the hydrolysis (activation) of oseltamivir. Oseltamivir is the most commonly prescribed drug for the treatment and prophylaxis of both influenza virus A and B infections, and proven effective in the treatment of the H1N1 2009 viral infection. However, drug resistance and sometimes severe side effects have been documented during oseltamivir therapy, though the underlying mechanisms remain incompletely understood. We hypothesize that impaired hCES1 function resulting from CES1 genetic polymorphisms is a significant and overlooked contributing factor to drug resistance and side effects reported with oseltamivir. In the present project, in vitro activation (Specific Aim 1) and antiviral effects (Specific Aim 2) of oseltamivir will be studied in the cell lines transfected with selected nonsynonymous CES1 variants. Additionally, a newly developed Ces1 knockout mouse model will be utilized to study the impact of aberrant CES1 on the activation and antiviral activity of oseltamivir in vivo (Specific Aim 3). Elucidating the correlation between CES1 genetic polymorphisms and the therapeutic outcomes of oseltamivir will permit a better understanding of the causes of individual variability in response and tolerability during oseltamivir therapy. Further, it may allow for the identification of subjects likely to be non-responders in advance of a therapeutic course of the drug. Through achievement of the Specific Aims, future research can be directed at both normal volunteer studies as well as larger clinical studies of oseltamivir in the treatment of influenza which incorporate CES1 genotyping, and evaluation of specific variants on therapeutic outcomes. Finally, the project holds the potential to improve the therapeutic efficacy and safety of many other medications metabolized (activated or deactivated) by hCES1.